The goal of the project is the search for new effective radiomitigators based on new understanding of mechanisms of radiation damage realized via mitochondria-dependent pathways. Based on our discovery of a new stage of apoptotic program that utilizes reactive oxygen species to cause peroxidation of a mitochondria-specific phospholipid (PL), cardiolipin (CL) by cytochrome c our central hypothesis is that small molecules causing suppression of irradiation induced peroxidation of CL in mitochondria - catalyzed within the complex of cyt c with CL - represent new types of radiomitigators. Unique interactions of cyt c with CL and features of the catalytic complex offer several potentially promising targets for the regulation of CL peroxidation that will be explored in the current proposal and represent its specific aims as follows: Specific Aim 1 will determine specific mechanisms defining development of CL peroxidation in cells and radiosensitive tissues of irradiated animals. Specific Aim 2 will develop and optimize small molecule inhibitors of cyt c/CL peroxidase complex and investigate mechanisms of their action to achieve maximized radiomitigating efficiency in cells and tissues. We plan to explore three novel mechanism-based types of candidate molecules: 1) N-heterocyle-substiuted fatty acids, 2) imidazole-substituted homologues of tocopherols (vitamin E), and 3) nitroxide-oximes. Specific Aim 3 will identify mechanism-based strategies and small molecules capable of regulating CL oxidizability and localization in mitochondria to limit its oxidation during irradiation induced apoptosis. This will be achieved by three different approaches: 1) by delivery into mitochondria of chemically modified CLs that can act as decoys to protect endogenous CL from oxidation, 2) by affecting CL biosynthesis to shift its molecular speciation in favor of non-oxidizable mono-unsaturated types of CL, and 3) by inhibiting mitochondrial scramblase-3 to prevent transmembrane redistribution of CL during radiation induced apoptosis. Overall, our proposed project will explore and optimize six new classes of anti-apoptotic small molecule regulators that may be used for the development of safe synthetic radiomitigators.